Devices for the detection of multiple analytes in a sample

ABSTRACT

The present invention relates generally to an assay for detecting and differentiating multiple analytes, if present, in a single fluid sample, including devices and methods therefore.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/211,253 filed Mar. 14, 2014, now U.S. Pat. No. 9,207,241, issuedon Dec. 8, 2015, which is continuation application of U.S. applicationSer. No. 12/718,739, filed Mar. 5, 2010, now U.S. Pat. No. 8,722,426,issued on May 13, 2014, which is a divisional of U.S. application Ser.No. 12/115,416, filed May 5, 2008, now U.S. Pat. No. 7,691,644, issuedon Apr. 6, 2010, which is a divisional of U.S. application Ser. No.10/948,358, filed Sep. 22, 2004, now U.S. Pat. No. 7,378,285, issued onMay 27, 2008, which claims priority benefit of U.S. ProvisionalApplication No. 60/505,092, filed Sep. 22, 2003. The contents of theseapplications are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to an assay for detecting anddifferentiating multiple analytes, if present, in a single fluid sample,including devices and methods therefor.

BACKGROUND OF THE INVENTION

The field of rapid diagnostic testing has evolved for many years topermit the detection of analytes in a variety of sample types. The useof polyclonal antibodies was followed by the use of monoclonalantibodies to generate assays with high specificity for a number ofanalytes, including hormones, blood cells, drugs and their metabolites,as well as the antigens of infectious agents including Strep A, Strep B,Chlamydia, HIV, RSV, influenza A and influenza B and many others. Thevisible signal generated by enzyme-catalyzed reactions or by theaccumulation of a visible signal at the level of a test line has alsoresulted in rapid development of highly sensitive results. Many of therapid immunoassay-based tests include a solid housing encasing a teststrip. However, recently, immunochromatographic assays have beenmanufactured which do not have solid housings. Such tests, referred toas dipsticks, can be dipped directly into a tube containing apre-determined amount of the liquid sample of interest. The extremity ofthe dipstick containing a sample-receiving pad is generally brought incontact with a liquid sample, and the liquid migrates up the flow path.Advantages of the dipstick format include ease of use and minimumhandling, which reduces the opportunities for contamination andprocedural errors, and lowers manufacturing costs.

One disadvantage of current immunochromatographic dipsticks is that theycan only detect the presence of a single analyte. Often these devicesare limited because there is no provision to mark the location ofpossible multiple test lines along the flow path. In the field ofchemical urinalysis, dipsticks carrying multiple pads, each specific fora urine analyte to be detected and measured, the dipstick is dipped intothe urine sample, then removed from the container, blotted to eliminateexcess urine, and applied against a template in order to read theresults. These devices are capable of evaluating multiple analytes, butare problematic. For example, such devices increase the chances ofcontamination by carry-over of material from one device to another, withthe consequence of potentially inaccurate results. Moreover, this formatexposes the user to potential contamination via removing the strip fromthe urine sample, blotting it on an absorbent paper, which becomescontaminated, applying it against the template, which often is theexterior wall of the product container, thereby contaminating theproduct package itself. Further, as indicated, an external template isrequired to read results.

It is recognized in a variety of fields that the use of single analyterapid tests is often limiting, for example, because only one analyte ata time can be evaluated. The advantage of rapidity is thereforechallenged by the limitation of current assays as adjuncts to thediagnosis of a disease state. For instance, the pediatric units have tomake differential diagnostics of Flu A, Flu B, RSV and other upperrespiratory viruses, on infants that are in need of urgent care. Theavailability of rapid test panels would greatly facilitate the doctors'efforts to diagnose the condition, and therefore to take the appropriatecourse of action faster, more easily, and at lower cost. To date, nosuch assay has been developed that allows the differential diagnostic oftwo or more analytes on a single test strip, in a minimally involvingprocedure.

In summary, chemical urinalysis dipstick assays have been used for manyyears to determine the presence (or amount) of multiple analytes in aurine sample; however, the technology used to perform such assays notonly has undesirable use characteristics but is not readily transferableto immunologic based assays (dipstick or lateral flow) which requireflow of sample through the assay device rather than immersion of thedevice (in particular immersion of the test portion of the device) intoa sample. For example, with respect to the use of chemical urinalysisdipsticks, the fact that they must be submerged into the urine sample,removed and blotted of excess urine then placed in physical contact (orvery close proximity) with an external, typically reusable (and hencecontaminable), test results panel (i.e., template) is not justundesirable but unsafe, particularly if the sample contains contagiousagents such as virus or bacteria. Further, because immunologic basedassays typically employ at least two, generally sequential reactions(for example a labeling followed by a capture (test) reaction), they arenot amenable to submersion into a sample in the same manner as thechemical urinalysis dipsticks. Thus, there is a need in the art fordevices and methods that addresses these problems in the art. Thepresent invention addresses these and other related needs in the art.

SUMMARY OF INVENTION

The present invention provides analytical devices, particularlyimmunoassay devices, capable of determining the presence and/or amountof multiple analytes in a fluid sample, permitting more completediagnosis or analysis of said sample. Advantageously, the presentdevices may be formatted as dipsticks or lateral flow devices, in eithercase not requiring an external test results panel for determination oftest results. By positioning the multiple test and/or control zones inpredetermined patterns on the test devices in accordance with thepresent invention, the need for an external test results panel, or evenmarkings on a test device housing, is eliminated. By way of example, ina preferred embodiment, a single control zone is positioned between twotest zones, each test zone testing for the presence or amount of adifferent analyte. It has heretofore been assumed that a control zonemust be located downstream of a test zone in order function as controlzone. However, the present inventors have discovered that such is notnecessary and, rather, that one or more control zones may be employed inimmunoassay devices not only to provide an indication of assaycompletion and/or operability but also of the relative location of oneor more test lines, thereby permitting rapid differentiation of analyteswithin a fluid sample in a single immunoassay test device.

In an embodiment of the present disclosure, a device is provided for thedetection of multiple analytes in a fluid sample, which devicecomprises: a matrix defining an axial flow path, the matrix comprising:i) a sample receiving zone at an upstream end of the flow path thatreceives the fluid sample, ii) a label zone positioned within the flowpath and downstream from the sample receiving zone, said label zonecomprising one or more labeled reagents which are capable of binding oneor more analytes to form labeled analytes and are mobilizable in thepresence of fluid sample, iii) one or more test zones positioned withinthe flow path and downstream from the label zone, wherein each of theone or more test zones contain means which permit the restraint of adifferent labeled analyte in each test zone or a combination ofdifferent labeled analyte in a single test zone, and wherein restrainedlabeled analyte is detectable within each test zone, and iv) one or morecontrol zones positioned within the flow path and downstream from thelabel zone, wherein the one or more control zones incorporate meanswhich permit the indication of the completion of an assay. In the mostfrequent embodiments, the device incorporates means to restrain andthereby detect two or more different labeled analytes in a sample. Also,in frequent embodiments, the device comprises a dipstick assay device.In occasional embodiments, the device comprises two or three or moretest zones and two or more control zones.

The present device permits the detection of multiple analytes in asample without reference to an external template. Moreover, frequentlythe device comprises a dipstick assay that lacks an external housing. Ingeneral, the analytes comprise analytes of interest and further comprisethose provided herein, among others. Frequently, the present devices areuseful for assaying a particular panel of analytes. Also frequently, thepresent devices are useful to simultaneously detect two or moredifferent analytes in a sample. On occasion the present device andmethods are useful to detect a panel of analytes of interest selectedfrom an influenza panel (comprising test zones containing reagentscapable of restraining a selection of influenza A, influenza B,respiratory syncytial virus (RSV), adenovirus, rhinovirus and/orparainfluenza virus), a panel comprising one or more of streptococcuspneumoniae, mycoplasma pneumoniae and/or Chlamydia, an HIV panel, aLupus panel, an H. Pylori panel, a toxoplasma panel, a herpes panel, aBorrelia panel, a rubella panel, a cytomegalovirus panels, a rheumatoidarthritis panel, or an Epstein-Barr panel, among others.

In one preferred aspect. each of the one or more test zones lie in fluidcommunication with one another. Moreover, in another aspect, the one ormore test zones lie in fluid communication with one or more controlzones. In a further aspect, the presently contemplated devices do notutilize one or a plurality of wells, rather a matrix defining an axialflow path is utilized. In a frequent embodiment, a device in accordancewith the present disclosure contains a single sample receiving zone thatlies in fluid communication with the one or more test zones.

In frequent embodiments, the control zone is positioned between the oneor more test zones. In occasional embodiments, the positioning of thecontrol zone between the one or more test zones comprises positioningone control zone between two test zones. Also in occasional embodiments,the positioning of the control zone between the one or more test zonescomprises multiple test zones and multiple control zones, wherein eachcontrol zone is positioned between two test zones, in an alternatingarrangement. Frequently, the control zone is positioned upstream of atest zone. Also frequently, the control zone is positioned downstream ofa test zone. In occasional embodiments, the control zone is positioneddownstream of each or all of the one or more test zones. In anotherembodiment, the test and control zones are positioned in an alternatingformat within the flow path beginning with a test zone positionedupstream of any control zone.

In one embodiment, each of the one or more test zones contain meanscomprising an immobilized reagent capable of specifically binding aunique analyte. Thus, in this embodiment, each of the one or more testzones contain an immobilized reagent capable of specifically binding aparticular analyte, wherein each of the immobilized reagents is capableof binding a different analyte than any other immobilized reagent withinanother test zone in the device. These means can comprise any of avariety of specific binding pair members as described elsewhere herein.On occasion, the means which permit the restraint of a different labeledanalyte in each test zone comprise an immobilized capture reagent.

In one aspect, the test zones can be provided in any of a variety shapesand configurations with the limitation that each particular test zone isdetectably distinguishable from other test zones, if present, and thecontrol zone(s) in the presence of labeled analyte, if present,restrained in that test zone. In a related aspect, the control zones canbe provided in any of a variety shapes and configurations with thelimitation that each particular control zone is detectablydistinguishable from other control zones, if present, and the test zonesupon completion of an assay.

In another embodiment, the label zone comprises multiple labeledreagents, wherein each of the multiple labeled reagents is capable ofspecifically binding a unique analyte. In occasional embodiments, thelabeled reagent is a reagent capable of binding any or all of themultiple analytes, if present, in the sample. Frequently, each of thelabeled reagents is detectably distinguishable from one another. Alsofrequently, the label component of the labeled reagent is selected fromthe group consisting of a chemiluminescent agent, a particulate label, acolloid label, a colorimetric agent, an energy transfer agent, anenzyme, a fluorescent agent and a radioisotope. In occasionalembodiments, the labeled reagents comprise different colored labeledreagents. For example, the labeled reagent can comprise 2, 3, 4, 5, or 6or more different colored particulate reagents. Particulate label colorscomprising red, blue, black, purple, and other high-contrast colors arefrequently utilized in the present embodiments. In frequent embodiments,the colored particulate label comprises a colored latex particulatelabel. In another frequent embodiment, the colored label comprises aCarbon, Gold or Selenium colored colloid label. On occasion, the use ofdifferent colored particulate labeled reagents allows for the detectionof multiple analytes via the observation of different detectable signals(e.g., different colors) in any one or more of the one or more testzones as a result of the restraint of different labeled analyte in eachtest zone. In a less occasional embodiment, the labeled reagent cancomprise a mixture of any of a variety of detectable labeling schemes inone device such that each analyte can become labeled by a differentlabeled reagent to provide a different detectable signal.

In occasional embodiments, a test zone may contain one or moreimmobilized reagents capable of specifically binding a unique analyte,such that multiple labeled analytes may become restrained and detectiblein the test zone. In this embodiment there may be multiple analytes ofinterest for the device, however, frequently only one of these analytesis present, if at all, at one time in the fluid sample. Thus, multiplelabeled reagents are useful in this embodiment which are both analytespecific and detectably distinguishable (i.e., different colors). Forexample, a device of the present embodiment is capable of detectingInfluenza A or influenza B antigen, if present, in a single sample. Inthe case of influenza A, a red-colored labeled reagent that is capableof specifically binding influenza A may be used, which would result inthe development of a red-colored test zone after completion of the assayof a fluid sample containing influenza A analyte. In the case ofInfluenza B, a blue-colored labeled reagent that is capable ofspecifically binding influenza B may be used, which would result in thedevelopment of a blue-colored test zone after completion of the assay ofa fluid sample containing influenza B analyte. One of skill in the artwould recognize that the colors may be alternated and/or otherdetectibly distinguishable labeling means contemplated herein may beutilized. Frequently, such test zones can be deposited as single zonescontaining a mixture of capture reagent, or as adjacent zones of singlecapture reagents. On occasion, in the present embodiment, multipleanalytes of interest are present together in the fluid sample, whichthen become labeled subsequent to contact with the device and restrainedin the test zone. Thus, multiple labeled analytes may be restrained in asingle test zone.

In another embodiment, the sample receiving zone and the label zonecomprise separate components in fluid-flow contact. In a frequentembodiment, the one or more test zones and the control zone arepositioned within a test region. Moreover, frequently, the samplereceiving zone, label zone and the test region comprise separatecomponents in fluid-flow contact. Also frequently, the test regioncomprises nitrocellulose or other material suitable for immobilizationof test and control reagents, and/or is laminated on a plastic backingmaterial. On occasion, the matrix is positioned within a housingcomprising a support and optionally a cover, wherein the housingcontains a sample-receiving aperture and one or more observation ports.In occasional embodiments, the control zone comprises a mark that isdetectable within the test region when the test region is in a moiststate. In this embodiment the test region comprises a material that isopaque in a dry state and transparent in a moist state such that themark become visible as the liquid sample moistens the test region.

In a further embodiment, the device is capable of detecting influenza Aand/or influenza B, if present, in a single sample. In occasionalembodiments, each of the multiple analytes are selected from the groupconsisting of a toxin, an organic compound, a protein, a peptide, amicroorganism, a bacteria, a virus, an amino acid, a nucleic acid, acarbohydrate, a hormone, a steroid, a vitamin, a drug, an antibody, anda hapten.

In another further embodiment, the number of control zones isrepresented by the variable “n” and the number of test zones isrepresented by the variable “n+1,” and wherein the test zones andcontrol zones are positioned in a series comprising an alternatingformat, wherein the zone arranged at the most downstream position in theseries comprises a test zone. The variable “n” often refers to 2 testzones. However, on occasion, the variable “n” refers to between 2 toabout 5 test zones. Thus, on occasion, 2, 3, 4, 5 or more test zones arepositioned on the device. Frequently, each of the test zones permit therestraint of a different analyte. On occasion, a device is provided thatis capable of detecting a number of different analytes represented bythe variable “n,” wherein the number of control zones and the number ofdifferent analytes capable of being detected by the device are equal.Also on occasion, a device is provided that is capable of detecting anumber of different analytes represented by the variable “n+1,” whereinthe number of test zones and the number of different analytes capable ofbeing detected by the device are equal.

In frequent embodiments a device for the detection of multiple analytesin a fluid sample is provided, wherein the device comprises: a matrixdefining an axial flow path, the matrix comprising: i) a samplereceiving zone at an upstream end of the flow path that receives thefluid sample; ii) a label zone, within the flow path and downstream fromthe sample receiving zone, comprising a first and second labeledreagent, each of which specifically bind an analyte to form a labeledanalyte and are mobilizable in the presence of fluid sample; and iii) atest region comprising a first test zone, a second test zone and acontrol zone, wherein the control zone is positioned between the firstand second test zones within the flow path, wherein each of the firstand second test zones contain means which permit the detection of adifferent analyte in each test zone, and wherein the control zoneincorporates means which allow for the indication of the completion ofan assay. In occasional embodiments, the first zone is positionedupstream from the control zone within the flow path and the second zoneis positioned downstream from the control zone within the flow path.

In another embodiment, methods are provided for the detection of one ormore analytes in a fluid sample. For example, a method is provided forthe detection of multiple analytes in a fluid sample, comprising: i)contacting a device of the type described above with a fluid samplesuspected of containing one or more analytes, and wherein each of theone or more test zones in the device contains means which permit therestraint of a different labeled analyte or combination of labeledanalytes in each test zone; and ii) detecting one or more labeledanalytes restrained in the one or more test zones. Frequently the devicecomprises a dipstick-type device. In general, the analytes of interestcomprise those provided herein, among others. Frequently, the presentmethods are useful for assaying a particular panel of analytes. Alsofrequently, the present methods are useful to simultaneously detect twoor more different analytes in a sample. Commonly the present devices andmethods are utilized to diagnose a medical condition. Also commonly, thepresent devices and methods aid in guiding therapeutic decisions. Onoccasion, the present method steps may be practiced at differentlocations, and by different entities.

These and other features and advantages of the present invention will beapparent from the following detailed description, examples and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A-J) depicts various example configurations of the test andcontrol zones within the presently contemplated devices. The arrow ineach Figure indicates the general direction of fluid sample flow afterinitial contact with the device. The boxes containing vertical linesand/or the diagonal lines depict control zones, and the boxes containingcrosshatched lines depict test zones. FIG. 1(A-J) further depicts thesample receiving zone [1], the label zone [2] and the test region[3].The present devices are not intended to be limited to the aspectsindicated in the depicted embodiments, other configurations arecontemplated. Moreover, the depicted aspects are not necessarilypresented to scale.

FIG. 2 is a graph depicting the ratio of test zone and control zonesignals for different volumes of sample in exemplary devices.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.” Theuse of the phrase “one or more” herein does not alter this intendedmeaning for the terms “a” or “an.”

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from, e.g., infection or genetic defect, andcharacterized by identifiable symptoms.

As used herein the term “sample” refers to anything which may contain ananalyte for which an analyte assay is desired. The sample may be abiological sample, such as a biological fluid or a biological tissue.Examples of biological fluids include urine, blood, plasma, serum,saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus,amniotic fluid or the like. Biological tissues are aggregate of cells,usually of a particular kind together with their intercellular substancethat form one of the structural materials of a human, animal, plant,bacterial, fungal or viral structure, including connective, epithelium,muscle and nerve tissues. Examples of biological tissues also includeorgans, tumors, lymph nodes, arteries and individual cell(s).

“Fluid sample” refers to a material suspected of containing theanalyte(s) of interest, which material has sufficient fluidity to flowthrough an immunoassay device in accordance herewith. The fluid samplecan be used as obtained directly from the source or following apretreatment so as to modify its character. Such samples can includehuman, animal or man-made samples. The sample can be prepared in anyconvenient medium which does not interfere with the assay. Typically,the sample is an aqueous solution or biological fluid as described inmore detail below.

The fluid sample can be derived from any source, such as a physiologicalfluid, including blood, serum, plasma, saliva, sputum, ocular lensfluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid,and the like. Herein, fluid homogenates of cellular tissues such as, forexample, hair, skin and nail scrapings, meat extracts and skins offruits and nuts are also considered biological fluids. Pretreatment mayinvolve preparing plasma from blood, diluting viscous fluids, and thelike. Methods of treatment can involve filtration, distillation,separation, concentration, inactivation of interfering components, andthe addition of reagents. Besides physiological fluids, other samplescan be used such as water, food products, soil extracts, and the likefor the performance of industrial, environmental, or food productionassays as well as diagnostic assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample onceit is modified to form a liquid medium or to release the analyte. Theselection and pretreatment of biological, industrial, and environmentalsamples prior to testing is well known in the art and need not bedescribed further.

As used herein, the term “specifically binds” refers to the bindingspecificity of a specific binding pair. “Specific pair binding member”refers to a member of a specific binding pair, i.e., two differentmolecules wherein one of the molecules specifically binds with thesecond molecule through chemical or physical means. The two moleculesare related in the sense that their binding with each other is such thatthey are capable of distinguishing their binding partner from otherassay constituents having similar characteristics. The members of thespecific binding pair are referred to as ligand and receptor(antiligand), sbp member and sbp partner, and the like. A molecule mayalso be a sbp member for an aggregation of molecules; for example anantibody raised against an immune complex of a second antibody and itscorresponding antigen may be considered to be an sbp member for theimmune complex.

In addition to antigen and antibody specific binding pair members, otherspecific binding pairs include, as examples without limitation, biotinand avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence and an antibody specific for the sequence or theentire protein, polymeric acids and bases, dyes and protein binders,peptides and specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), metals and their chelators, and the like.Furthermore, specific binding pairs can include members that are analogsof the original specific binding member, for example an analyte-analogor a specific binding member made by recombinant techniques or molecularengineering.

An sbp member is analogous to another sbp member if they are bothcapable of binding to another identical complementary sbp member. Suchan sbp member may, for example, be either a ligand or a receptor thathas been modified by the replacement of at least one hydrogen atom by agroup to provide, for example, a labeled ligand or labeled receptor. Thesbp members can be analogous to or complementary to the analyte or to ansbp member that is complementary to the analyte.

If the specific binding member is an immunoreactant it can be, forexample, an antibody, antigen, hapten, or complex thereof. If anantibody is used, it can be a monoclonal or polyclonal antibody, arecombinant protein or antibody, a chimeric antibody, a mixture(s) orfragment(s) thereof, as well as a mixture of an antibody and otherspecific binding members. The details of the preparation of suchantibodies and their suitability for use as specific binding members areknown to those skilled in the art.

“Antigen” shall mean any compound capable of binding to an antibody, oragainst which antibodies can be raised.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant regions, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively. Typically, an antibody is animmunoglobulin having an area on its surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies or fragment thereof made by recombinantmethods.

“Analyte” refers to the compound or composition to be detected ormeasured and which has at least one epitope or binding site. The analytecan be any substance for which there exists a naturally occurringanalyte specific binding member or for which an analyte-specific bindingmember can be prepared. e.g., carbohydrate and lectin, hormone andreceptor, complementary nucleic acids, and the like. Further, possibleanalytes include virtually any compound, composition, aggregation, orother substance which may be immunologically detected. That is, theanalyte, or portion thereof, will be antigenic or haptenic having atleast one determinant site, or will be a member of a naturally occurringbinding pair.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof. A non-exhaustive list ofexemplary analytes is set forth in U.S. Pat. No. 4,366,241, at column19, line 7 through column 26, line 42, the disclosure of which isincorporated herein by reference. Further descriptions and listings ofrepresentative analytes are found in U.S. Pat. Nos. 4,299,916;4,275,149; and 4,806,311, all incorporated herein by reference.

“Labeled reagent” refers to a substance comprising a detectable labelattached with a specific binding member. The attachment may be covalentor non-covalent binding, but the method of attachment is not critical tothe present invention. The label allows the label reagent to produce adetectable signal that is related to the presence of analyte in thefluid sample. The specific binding member component of the label reagentis selected to directly bind to the analyte or to indirectly bind theanalyte by means of an ancillary specific binding member, which isdescribed in greater detail hereinafter. The label reagent can beincorporated into the test device at a site upstream from the capturezone, it can be combined with the fluid sample to form a fluid solution,it can be added to the test device separately from the test sample, orit can be predeposited or reversibly immobilized at the capture zone. Inaddition, the specific binding member may be labeled before or duringthe performance of the assay by means of a suitable attachment method.

“Label” refers to any substance which is capable of producing a signalthat is detectable by visual or instrumental means. Various labelssuitable for use in the present invention include labels which producesignals through either chemical or physical means. Such labels caninclude enzymes and substrates, chromogens, catalysts, fluorescentcompounds, chemiluminescent compounds, and radioactive labels. Othersuitable labels include particulate labels such as colloidal metallicparticles such as gold, colloidal non-metallic particles such asselenium or tellurium, dyed or colored particles such as a dyed plasticor a stained microorganism, organic polymer latex particles andliposomes, colored beads, polymer microcapsules, sacs, erythrocytes,erythrocyte ghosts, or other vesicles containing directly visiblesubstances, and the like. Typically, a visually detectable label is usedas the label component of the label reagent, thereby providing for thedirect visual or instrumental readout of the presence or amount of theanalyte in the test sample without the need for additional signalproducing components at the detection sites.

The selection of a particular label is not critical to the presentinvention, but the label will be capable of generating a detectablesignal either by itself, or be instrumentally detectable, or bedetectable in conjunction with one or more additional signal producingcomponents, such as an enzyme/substrate signal producing system. Avariety of different label reagents can be formed by varying either thelabel or the specific binding member component of the label reagent; itwill be appreciated by one skilled in the art that the choice involvesconsideration of the analyte to be detected and the desired means ofdetection. As discussed below, a label may also be incorporated used ina control system for the assay.

For example, one or more signal producing components can be reacted withthe label to generate a detectable signal. If the label is an enzyme,then amplification of the detectable signal is obtained by reacting theenzyme with one or more substrates or additional enzymes and substratesto produce a detectable reaction product.

In an alternative signal producing system, the label can be afluorescent compound where no enzymatic manipulation of the label isrequired to produce the detectable signal. Fluorescent moleculesinclude, for example, fluorescein, phycobiliprotein, rhodamine and theirderivatives and analogs are suitable for use as labels in such a system.

The use of dyes for staining biological materials, such as proteins,carbohydrates, nucleic acids, and whole organisms is documented in theliterature. It is known that certain dyes stain particular materialspreferentially based on compatible chemistries of dye and ligand. Forexample, Coomassie Blue and Methylene Blue for proteins, periodicacid-Schiff s reagent for carbohydrates, Crystal Violet, Safranin O, andTrypan Blue for whole cell stains, ethidium bromide and Acridine Orangefor nucleic acid staining, and fluorescent stains such as rhodamine andCalcofluor White for detection by fluorescent microscopy. Furtherexamples of labels can be found in, at least, U.S. Pat. Nos. 4,695,554;4,863,875; 4,373,932; and 4,366,241, all incorporated herein byreference.

“Signal producing component” refers to any substance capable of reactingwith another assay reagent or with the analyte to produce a reactionproduct or signal that indicates the presence of the analyte and that isdetectable by visual or instrumental means. “Signal production system”,as used herein, refers to the group of assay reagents that are needed toproduce the desired reaction product or signal.

“Observable signal” as used herein refers to a signal produced in theclaimed devices and methods that is detectable by visual inspection.Without limitation, the type of signal produced depends on the labelreagents and marks used (described herein). Generally, observablesignals indicating the presence or absence of an analyte in a sample maybe evident of their own accord, e.g., plus or minus signs orparticularly shaped symbols, or may be evident through the comparisonwith a panel such as a color indicator panel.

“Axial flow” as used herein refers to lateral, vertical or transverseflow through a particular matrix or material comprising one or more testand/or control zones. The type of flow contemplated in a particulardevice, assay or method varies according to the structure of the device.Without being bound by theory, lateral, vertical or transverse flow mayrefer to flow of a fluid sample from the point of fluid contact on oneend or side of a particular matrix (the upstream or proximal end) to anarea downstream (or distal) of this contact. The downstream area may beon the same side or on the opposite side of the matrix from the point offluid contact. For example, in vertical flow devices of the presentinvention, axial flow may progress vertically from and through a firstmember (top to bottom) to a second member and from there on to anabsorbent medium. By way of further example, and as will be appreciatedby those of skill in the art, in a vertical flow device configured, forexample, as a dipstick, a fluid sample may flow literally up the device,in which case however, the point of first contact of the fluid sample tothe device is nonetheless considered the upstream (i.e., proximal) endand the point of termination of flow the downstream (i.e., distal) end.

“Absorbent material” as used herein refers to material used in verticalflow devices and assays that allows and promotes sample flow through thefirst and second members. Such materials may be as described in, e.g.,U.S. Pat. No. 4,632,901, such as, for example, fibrous materials such ascellulose acetate fibers, cellulose or cellulose derivatives, polyester,or polyolefin. Generally, the absorbent material, as used herein, shouldmaintain direct or intimate contact with the second member in order topromote fluid flow therethrough. Contemplated absorbent materials havingfluid absorptive qualities are generally compressible and may becompressed in devices of the present invention to ensure contact withthe second member or positive control element.

As used herein the phrase “mark that is detectable within the testregion when the test region is in a moist state” refers to the type ofmark described, for example in U.S. patent application Ser. No.09/950,366, filed, Sep. 10, 2001, currently pending and published asU.S. Patent Application Publication No. 20030049167, and Ser. No.10/241,822, filed Sep. 10, 2002, currently pending and published as U.S.Patent Application Publication No. 20030157699.

As used herein the terms “upstream” and “downstream” refer to thedirection of fluid sample flow subsequent to contact of the fluid samplewith a representative device of the present disclosure, wherein, undernormal operating conditions, the fluid sample flow direction runs froman upstream position to a downstream position. For example, when fluidsample is initially contacted with the sample receiving zone, the fluidsample then flows downstream through the label zone and so forth.

As used herein the phrase “completion of an assay” refers to axial flowof applied liquid sample suspected of containing one or more analytesthrough a representative device, downstream of at least one test zoneand at least one control zone. Thus, as used herein multiple assayscould be completed in a single device which comprises multiple pairs ofalternating test and control zones. More commonly, the phrase completionof assay refers to axial flow of applied liquid sample suspected ofcontaining one or more analytes through a representative device,downstream of all test and control zones on or in the device.

B. Test Devices

The present description provides for the development and use of singleor multiple control zones in a single immunoassay device that arepositioned in a predetermined manner relative to individual test zonesthereby allowing easy identification of each of the one or more analytesof interest tested for in the device. The present description furtherprovides for the making of control zones of various shapes, physical orchemical identities, and colors. In part, the use of such control zonesallows for immunoassay devices, particularly including dipsticks, thatare easy to use, and allow for the identification of multiple analytesduring a single assay procedure.

The present description further provides means to build a rapid,multi-analyte assay, which is needed in many fields of environmentalmonitoring, medicine, particularly in the field of infectious disease.For example, contemplated devices include those useful for thedifferential diagnosis of Flu A or Flu B, which may result in differenttreatments, or the differential diagnosis of Flu A, Flu B, and/or RSV inone step. Such devices permit the use of a single sample for assayingmultiple analytes at once, and beneficially allows for a considerablereduction of the hands-on time and duration of the diagnostic processfor the benefit of the doctor, or user in general.

A variety of analytes may be assayed utilizing devices and methods ofthe present disclosure. In a particular device useful for assaying forone or more analytes of interest in a sample, the collection of analytesof interest may be referred to as a panel. For example, a panel maycomprise any combination (or all of) of influenza A, influenza B,respiratory syncytial virus (RSV), adenovirus, and parainfluenza virus.Another panel may comprise testing for a selection of one or more ofupper respiratory infection including, for example, streptococcuspneumoniae, mycoplasma and/or pneumoniae. Yet another panel can bedevised for the diagnosis of sexually transmitted disease including, forexample, Chlamydia, Trichomonas and/or Gonorrhea.

On occasion a panel may optionally include a variety of other analytesof interest, including SARS-associated coronavirus, influenza C; ahepatitis panel comprising a selection of hepatitis B surface Ag or Ab,hepatitis B core Ab, hepatitis A virus Ab, and hepatitis C virus; aphospholipids panel comprising a selection of Anticardiolipin Abs (IgG,IgA, and IgM Isotypes); an arthritis panel comprising a selection ofrheumatoid factor, antinuclear antibodies, and Uric Acid; an EpsteinBarr panel comprising a selection of Epstein Barr Nuclear Ag, EpsteinBarr Viral Capsid Ag, and Epstein Barr Virus, Early Antigen; otherpanels include HIV panels, Lupus panels, H. Pylori panels, toxoplasmapanels, herpes panels, Borrelia panels, rubella panels, cytomegaloviruspanels, and many others. One of skill in art would understand that avariety of panels may be assayed via the immunoassays described herein.See, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, John E. et. al.,eds. 1999).

Other fields of interest include the diagnosis of veterinary diseases,analysis of meat, poultry, fish for bacterial contamination, inspectionof food plants, restaurants, hospitals and other public facilities,analysis of environmental samples including water for beach, lakes orswimming pool contamination. Analytes detected by these tests includeviral and bacterial antigens as well as chemicals including, forexample, lead, pesticides, hormones, drugs and their metabolites,hydrocarbons and all kinds of organic or inorganic compounds.

The present disclosure provides a test device, particularly immunoassaydevices, for determining the presence or absence of multiple analytes ina fluid sample. In general, a test device of the present disclosureincludes a matrix defining an axial flow path. Typically, the matrixfurther includes a sample receiving zone, a label zone, a test zone anda control zone. In frequent embodiments, a test region comprises thetest and control zones. In a related embodiment, the matrix furtherincludes an absorbent zone disposed downstream of the test region.Moreover, in preferred embodiments, the test region, which comprises thetest and control zones, is observable.

Numerous analytical devices known to those of skill in the art may beadapted in accordance with the present invention, to detect multipleanalytes. By way of example, dipstick, lateral flow and flow-throughdevices, particularly those that are immunoassays, may be modified inaccordance herewith in order to detect and distinguish multipleanalytes. Exemplary lateral flow devices include those described in U.S.Pat. Nos. 4,818,677, 4,943,522, 5,096,837 (RE 35,306), 5,096,837,5,118,428, 5,118,630, 5,221,616, 5,223,220, 5,225,328, 5,415,994,5,434,057, 5,521,102, 5,536,646, 5,541,069, 5,686,315, 5,763,262,5,766,961, 5,770,460, 5,773,234, 5,786,220, 5,804,452, 5,814,455,5,939,331, 6,306,642. Other lateral flow devices that may be modifiedfor use in distinguishable detection of multiple analytes in a fluidsample include U.S. Pat. Nos. 4,703,017, 6,187,598, 6,352,862,6,485,982, 6,534,320 and 6,767,714. Exemplary dipstick devices includethose described in U.S. Pat. Nos. 4,235,601, 5,559,041, 5,712,172 and6,790,611. It will be appreciated by those of skill in the art that theaforementioned patents may and frequently do disclose more than oneassay configuration and are likewise referred to herein for suchadditional disclosures. Advantageously, the improvements described areapplicable to various assay, especially immunoassay, configurations.

In a frequent embodiment, the sample receiving zone accepts a fluidsample that may contain analytes of interest. In another embodiment, thesample receiving zone is dipped into a fluid sample. A label zone islocated downstream of the sample receiving zone, and contains one ormore mobile label reagents that recognize, or are capable of binding theanalytes of interest. Further, a test region is disposed downstream fromthe label zone, and contains test and control zones. The test zone(s)generally contain means which permit the restraint of a particularanalyte of interest in each test zone. Frequently, the means included inthe test zone(s) comprise an immobilized capture reagent that binds tothe analyte of interest. Generally the immobilized capture reagentspecifically binds to the analyte of interest. Although, on occasion,the means which permit the restraint of a particular analyte of interestin each test zone comprise another physical, chemical or immunologicalmeans for specifically restraining an analyte of interest. Thus, as thefluid sample flows along the matrix, the analyte of interest will firstbind with a mobilizable label reagent in the label zone, and then becomerestrained in the test zone. In occasional embodiments, the test regionis comprised of a material that is opaque in a dry state and transparentin a moist state. Thus, when a control zone comprising a mark on thedevice is utilized, this mark is positioned about the test region suchthat it becomes visible within the test region when the test region isin a moist state.

In another preferred embodiment, the fluid sample flows along a flowpath running from the sample receiving zone (upstream), through thelabel zone, and then to the test and control zones (together comprisedin a test region) (downstream). Optionally, the fluid sample maythereafter continue to the absorbent zone.

In one embodiment, the sample receiving zone is comprised of anabsorbent application pad. Suitable materials for manufacturingabsorbent application pads include, but are not limited to, hydrophilicpolyethylene materials or pads, acrylic fiber, glass fiber, filter paperor pads, desiccated paper, paper pulp, fabric, and the like. Forexample, the sample receiving zone may be comprised of a material suchas a nonwoven spunlaced acrylic fiber, i.e., New Merge (available fromDuPont) or HDK material (available from HDK Industries, Inc.). In arelated embodiment, the sample receiving zone is constructed from anymaterial that is capable of absorbing water.

In another embodiment, the sample receiving zone is comprised of anymaterial from which the fluid sample can pass to the label zone.Further, the absorbent application pad can be constructed to act as afilter for cellular components, hormones, particulate, and other certainsubstances that may occur in the fluid sample. Application pad materialssuitable for use by the present invention also include those applicationpad materials disclosed in U.S. Pat. No. 5,075,078, incorporated hereinby reference.

The functions of the sample receiving zone may include, for example: pHcontrol/modification and/or specific gravity control/modification of thesample applied, removal or alteration of components of the sample whichmay interfere or cause non-specific binding in the assay, or to directand control sample flow to the test region. The filtering aspect allowsan analyte of interest to migrate through the device in a controlledfashion with few, if any, interfering substances. The filtering aspect,if present, often provides for a test having a higher probability ofsuccess and accuracy. In another embodiment, the sample receiving zonemay also incorporate reagents useful to avoid cross-reactivity withnon-target analytes that may exist in a sample and/or to condition thesample; depending on the particular embodiment, these reagents mayinclude non-hCG blockers, anti-RBC reagents, Tris-based buffers, EDTA,among others. When the use of whole blood is contemplated, anti-RBCreagents are frequently utilized. In yet another embodiment, the samplereceiving zone may incorporate other reagents such as ancillary specificbinding members, fluid sample pretreatment reagents, and signalproducing reagents.

In a further embodiment, the sample receiving zone is comprised of anadditional sample application member (e.g., a wick). Thus, in oneaspect, the sample receiving zone can comprise a sample application padas well as a sample application member. Often the sample applicationmember is comprised of a material that readily absorbs any of a varietyof fluid samples contemplated herein, and remains robust in physicalform. Frequently, the sample application member is comprised of amaterial such as white bonded polyester fiber. Moreover, the sampleapplication member, if present, is positioned in fluid-flow contact witha sample application pad. This fluid flow contact can comprise anoverlapping, abutting or interlaced type of contact. In occasionalembodiments, the sample application member may be treated with ahydrophilic finishing. Often the sample application member, if present,may contain similar reagents and be comprised of similar materials tothose utilized in exemplary sample application pads.

In another embodiment, the test device is configured to perform animmunological analysis process. In yet another embodiment, the liquidtransport along the matrix is based upon capillary action. In a furtherembodiment, the liquid transport along the matrix is based onnon-bibulous lateral flow, wherein all of the dissolved or dispersedcomponents of the liquid sample are carried at substantially equal ratesand with relatively unimpaired flow laterally through the matrix, asopposed to preferential retention of one or more components as wouldoccur, e.g., in materials that interact, chemically, physically,ionically or otherwise with one or more components. See for example,U.S. Pat. No. 4,943,522, hereby incorporated by reference in itsentirety.

One purpose of the label zone is to maintain label reagents and controlreagents in a stable state and to facilitate their rapid and effectivesolubilization, mobilization and specific reaction with analytes ofinterest potentially present in a fluid sample.

In one embodiment, the label zone is comprised of a porous material suchas high density polyethylene sheet material manufactured by PorexTechnologies Corp. of Fairburn, Ga., USA. The sheet material has an openpore structure with a typical density, at 40% void volume, of 0.57 gm/ccand an average pore diameter of 1 to 250 micrometers, the averagegenerally being from 3 to 100 micrometers. In another embodiment, thelabel zone is comprised of a porous material such as a nonwovenspunlaced acrylic fiber (similar to the sample receiving zone), e.g.,New Merge or HDK material. Often, the porous material may be backed by,or laminated upon, a generally water impervious layer, e.g., Mylar. Whenemployed, the backing is generally fastened to the matrix by an adhesive(e.g., 3M 444 double-sided adhesive tape). Typically, a water imperviousbacking is used for membranes of low thickness. A wide variety ofpolymers may be used provided that they do not bind nonspecifically tothe assay components and do not interfere with flow of the fluid sample.Illustrative polymers include polyethylene, polypropylene, polystyreneand the like. On occasion, the matrix may be self-supporting. Othermembranes amenable to non-bibulous flow, such as polyvinyl chloride,polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride,polyamide, polycarbonate, polystyrene, and the like, can also be used.In yet another embodiment, the label zone is comprised of a materialsuch as untreated paper, cellulose blends, nitrocellulose, polyester, anacrylonitrile copolymer, and the like. The label zone may be constructedto provide either bibulous or non-bibulous flow, frequently the flowtype is similar or identical to that provided in at least a portion ofthe sample receiving zone. In a frequent embodiment, the label zone iscomprised of a nonwoven fabric such as Rayon or glass fiber. Other labelzone materials suitable for use by the present invention include thosechromatographic materials disclosed in U.S. Pat. No. 5,075,078, which isherein incorporated by reference.

In a frequent embodiment, the label zone material is treated withlabeled solution that includes material-blocking and label-stabilizingagents. Blocking agents include bovine serum albumin (BSA), methylatedBSA, casein, nonfat dry milk. Stabilizing agents are readily availableand well known in the art, and may be used, for example, to stabilizelabeled reagents. In frequent embodiments, employment of the selectedblocking and stabilizing agents together with labeled reagent in thelabeling zone followed by the drying of the blocking and stabilizingagents (e.g., a freeze-drying or forced air heat drying process) isutilized to attain improved performance of the device.

The label zone generally contains a labeled reagent, often comprisingone or more labeled reagents. In many of the presently contemplatedembodiments, multiple types of labeled reagents are incorporated in thelabel zone such that they may permeate together with a fluid samplecontacted with the device. These multiple types of labeled reagent canbe analyte specific or control reagents and may have differentdetectable characteristics (e.g., different colors) such that onelabeled reagent can be differentiated from another labeled reagent ifutilized in the same device. As the labeled reagents are frequentlybound to a specific analyte of interest subsequent to fluid sample flowthrough the label zone, differential detection of labeled reagentshaving different specificities (including analyte specific and controllabeled reagents) may be a desirable attribute. However, frequently, theability to differentially detect the labeled reagents having differentspecificities based on the label component alone is not necessary due tothe presence of defined test and control zones in the device, whichallow for the accumulation of labeled reagent in designated zones.

In one embodiment, a nonparticulate labeling scheme is contemplated. Inthese devices, a label which is a dyed antibody-enzyme complex isutilized. This dyed antibody-enzyme complex can be prepared bypolymerizing an antibody-enzyme conjugate in the presence of enzymesubstrate and surfactant. See, e.g., WO 9401775. Generally, the labelzone contains detectible moieties comprising enzyme-antibody conjugate,particulate labeled reagents, or dye labeled reagents, metal sol labeledreagents, etc., or moieties which may or may not be visible, but whichcan be detected if accumulated in the test and/or control zones. Thedetectible moieties can be dyes or dyed polymers which are visible whenpresent in sufficient quantity, or can be, and are preferred to beparticles such as dyed or colored latex beads, liposomes, metallic ornon-metallic colloids, organic, inorganic or dye solutions, dyed orcolored cells or organisms, red blood cells and the like. The detectiblemoieties used in the assay provide the means for detection of the natureof and/or quantity of result, and accordingly, their localization in thetest zones may be a function of the analyte in the sample. In general,this can be accomplished by coupling the detectible moieties to a ligandwhich binds specifically to an analyte of interest, or which competeswith an analyte of interest for the means which permit the restraint ofan analyte of interest positioned in the test zone(s). In the firstapproach, the detectible moieties are coupled to a specific bindingpartner which binds the analyte specifically. For example, if theanalyte is an antigen, an antibody specific for this antigen may beused; immunologically reactive fragments of the antibody, such asF(ab′)2, Fab or Fab′ can also be used. These ligands coupled to thedetectible moieties then bind to an analyte of interest if present inthe sample as the sample passes through the labeling zone and arecarried into the test region by the fluid flow through the device. Whenthe labeled analyte reaches the capture zone, it is restrained by arestraint reagent which is analyte-specific, label/detectiblemoiety-specific, or ligand-specific, such as an antibody or anothermember of a specific binding pair. In the second approach, the conjugateor particulate moieties are coupled to a ligand which is competitivewith analyte for an analyte-specific restraint reagent in a test zone.Both the analyte from the sample and the competitor bound to thedetectible moieties progress with the flow of the fluid sample to thetest region. Both analyte and its competitor then react with theanalyte-specific restraint reagent positioned in a test zone. Theunlabeled analyte thus is able to reduce the quantity ofcompetitor-conjugated detectible moieties which are retained in the testzone. This reduction in retention of the detectible moieties becomes ameasure of the analyte in the sample.

The labeling zone of immunoassay devices of the present invention mayalso include control-type reagents. These labeled control reagents oftencomprise detectible moieties that will not become restrained in the testzones and that are carried through to the test region and controlzone(s) by fluid sample flow through the device. In a frequentembodiment, these detectible moieties are coupled to a member of aspecific binding pair to form a control conjugate which can then berestrained in a separate control zone of the test region by acorresponding member of the specific binding pair to verify that theflow of liquid is as expected. The visible moieties used in the labeledcontrol reagents may be the same or different color, or of the same ordifferent type, as those used in the analyte of interest specificlabeled reagents. If different colors are used, ease of observing theresults may be enhanced. Generally, as used herein, the labeled controlreagents are also referred to herein together with analyte specificlabeled reagents or labeled test reagents as “labeled reagent(s).”

The test region is frequently comprised of a material such as cellulose,nitrocellulose, nylon, or hydrophilic polyvinylidene difluoride (PVDF).Hydrophilic polyvinylidene difluoride (PVDF) (available from Millipore,Billerica, Mass.). The term “nitrocellulose” is meant any nitric acidester of cellulose. Thus suitable materials may include nitrocellulosein combination with carboxylic acid esters of cellulose. The pore sizeof nitrocellulose membranes may vary widely, but is frequently withinabout 5 to 20 microns, preferably about 8 to 15 microns. However, othermaterials are contemplated which are known to those skilled in the art.In a frequent embodiment, the test region comprises a nitrocellulose webassembly made of Millipore nitrocellulose roll laminated to a clearMylar backing. In another embodiment, the test region is made of nylon.In less occasional embodiment, the test region is comprised of amaterial that can immobilize latex or other particles which carry asecond reagent capable of binding specifically to an analyte, therebydefining a test zone, for example, compressed nylon powder, or fiberglass. In an occasional embodiment, the test region is comprised of amaterial that is opaque when in a dry state, and transparent when in amoistened state. Preferably, the test and control zones may beconstructed from any of the materials as listed above for the testregion. Often the test and control zones form defined components of thetest region. In a particularly preferred embodiment, the test andcontrol zones are comprised of the same material as the test region.Frequently, the term “test region” is utilized herein to refer to aregion in/on a device that comprises at least the test and controlzones. To provide non-bibulous flow, these materials may be andpreferably are treated with blocking agents that can block the forceswhich account for the bibulous nature of bibulous membranes. Suitableblocking agents include bovine serum albumin, methylated bovine serumalbumin, whole animal serum, casein, and non-fat dry milk, as well as anumber of detergents and polymers, e.g., PEG, PVA and the like.Preferably the interfering sites on the untreated bibulous membranes arecompletely blocked with the blocking agent to permit non-bibulous flowthere through. As indicated herein, the present disclosure envisages atest device with multiple test and control zones.

The test region generally includes a control zone that is useful toverify that the sample flow is as expected. Each of the control zonescomprise a spatially distinct region that often includes an immobilizedmember of a specific binding pair which reacts with a labeled controlreagent. In an occasional embodiment, the procedural control zonecontains an authentic sample of the analyte of interest, or a fragmentthereof. In this embodiment, one type of labeled reagent can beutilized, wherein fluid sample transports the labeled reagent to thetest and control zones; and the labeled reagent not bound to an analyteof interest will then bind to the authentic sample of the analyte ofinterest positioned in the control zone. In another embodiment, thecontrol line contains antibody that is specific for, or otherwiseprovides for the immobilization of, the labeled reagent. In operation, alabeled reagent is restrained in each of the one or more control zones,even when any or all the analytes of interest are absent from the testsample.

In a less occasional embodiment, a labeled control reagent is introducedinto the fluid sample flow, upstream from the control zone. For example,the labeled control reagent may be added to the fluid sample before thesample is applied to the assay device. In frequent embodiments, thelabeled control reagent may be diffusively bound in the sample receivingzone, but is preferably diffusively bound in the label zone.

Exemplary functions of the labeled control reagents and zones include,for example, the confirmation that the liquid flow of the sampleeffectively solubilized and mobilized the labeled reagents deposited inthe label zone, that a sufficient amount of liquid traveled correctlythrough the sample receiving zone, label zone, and the test and controlzones, such that a sufficient amount of analyte could react with thecorresponding specific label in the label zone, migrate onto the testregion comprising the test and control zones, cross the test zone(s) inan amount such that the accumulation of the labeled analyte wouldproduce a visible or otherwise readable signal in the case of a positivetest result in the test zone(s). Moreover, an additional function of thecontrol zones may be to act as reference zones which allow the user toidentify the test results which are displayed as readable zones.

Since the devices of the present invention may incorporate one or morecontrol zones, the labeled control reagent and their correspondingcontrol zones are preferably developed such that each control zone willbecome visible with a desired intensity for all control zones afterfluid sample is contacted with the device, regardless of the presence orabsence of one or more analytes of interest.

In one embodiment, a single labeled control reagent will be captured byeach of the control zones on the test strip. Frequently, such a labeledcontrol reagent will be deposited onto or in the label zone in an amountexceeding the capacity of the total binding capacity of the combinedcontrol zones if multiple control zones are present. Accordingly, theamount of capture reagent specific for the control label can bedeposited in an amount that allows for the generation of desired signalintensity in the one or more control zones, and allows each of thecontrol zones to restrain a desired amount of labeled control reagent.At the completion of an assay, each of the control zones preferablyprovide a desired and/or pre-designed signal (in intensity and form).Examples of contemplated pre-designed signals include signals of equalintensities in each control zone, or following a desired pattern ofincreasing, decreasing or other signal intensity in the control zones.

In another embodiment, each control zone will be specific for a uniquecontrol reagent. In this embodiment, the label zone may include multipleand different labeled control reagents, equaling the number of controlzones in the assay, or a related variation. Wherein each of the labeledcontrol reagents may become restrained in one or more pre-determined andspecific control zone(s). These labeled control reagents can provide thesame detectible signal (e.g., be of the same color) or providedistinguishable detectible signals (e.g., have different colored labelsor other detection systems) upon accumulation in the control zone(s).

In yet another embodiment, the control zones may include a combinationof the two types of control zones described in the two previousembodiments, specifically, one or more control zones are able torestrain or bind a single type of labeled control reagent, and othercontrol zones on the same test strip will be capable of binding one orseveral other specific labeled control reagents.

In one embodiment, the labeled control reagent comprises a detectiblemoiety coupled to a member of a specific binding pair. Typically, alabeled control reagent is chosen to be different from the reagent thatis recognized by the means which are capable of restraining an analyteof interest in the test zone. Further, the labeled control reagent isgenerally not specific for the analyte. In a frequent embodiment, thelabeled control reagent is capable of binding the corresponding memberof a specific binding pair or control capture partner that isimmobilized on or in the control zone. Thus the labeled control reagentis directly restrained in the control zone.

In another embodiment, the detectable moiety which forms the labelcomponent of the labeled control reagent is the same detectible moietyas that which is utilized as the label component of the analyte ofinterest labeled test reagent. In a frequent embodiment, the labelcomponent of the labeled control reagent is different from the labelcomponent of the labeled test reagent, so that results of the assay areeasily determined. In another frequent embodiment, the control label andthe test label include colored beads, e.g., colored latex. Alsofrequently, the control and test latex beads comprise different colors.

In a further embodiment, the labeled control reagent includesstreptavidin, avidin or biotin and the control capture partner includesthe corresponding member of such specific binding pairs, which readilyand specifically bind with one another. In one example, the labeledcontrol reagent includes biotin, and the control capture partnerincludes streptavidin. The artisan will appreciate that other members ofspecific binding pairs can alternatively be used, including, forexample, antigen/antibody reactions unrelated to analyte.

The use of a control zone is helpful in that appearance of a signal inthe control zone indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in a test zonecan be noted.

In still further embodiment, a control zone comprising a mark thatbecomes visible in the test region when the test regions is in a moiststate is utilized. Control zones of this type are described in U.S.patent application Ser. No. 09/950,366, filed, Sep. 10, 2001, currentlypending and published as U.S. Patent Application Publication No.20030049167, and Ser. No. 10/241,822, filed Sep. 10, 2002, currentlypending and published as U.S. Patent Application Publication No.20030157699.

In occasional embodiments, one or more control zones of this type areutilized. In another embodiment, a combination of control zones of thetype utilizing labeled control reagents and control zone and of the typethat display the control zone when in a moist state can be used. Thisallows a simple way to formulate control zones while allowing to use areagent-based control zone to ascertain that the re-solubilization andmobilization of the reagents in the label pad process has beeneffective, and that the specific reactions took place as expected, allalong the path defined by the sample pad, label pad, test strip andabsorbent pad. The present embodiment includes the use of one or morecontrol zones that become visible when the test region is in the moiststate for each of the control zones of an assay, except the control zoneon the distal or downstream end of the test strip.

As indicated above, labeled test reagents are further provided whichfrequently comprise a test label coupled to a member of a specificbinding pair that is capable of specifically binding an analyte ofinterest. Thus, in general, multiple labeled test reagents arepositioned in the label zone, each of which is specific for apredetermined analyte of interest.

Test zones of the present description include means that permit therestraint of an analyte of interest. Frequently, test zones of thepresent description include a ligand that is capable of specificallybinding to an analyte of interest. Alternatively, test zones of thepresent description include a ligand that is capable of specificallybinding the labeled reagent bound to an analyte of interest. Inpractice, a labeled test reagent binds an analyte of interest present ina fluid sample after contact of the sample with a representative deviceand flow of the fluid sample into and through the label zone.Thereafter, the fluid sample containing the labeled analyte progressesto a test zone and becomes restrained in the test zone. The accumulationof labeled analyte in the test zone produces a detectible signal.Frequently, devices of the present disclosure incorporate one or moretest zones, each of which is capable of restraining different analytes,if present, in a fluid sample. Thus, in representative embodiments two,three, four, five or more (labeled) analytes of interest can berestrained in a single or different test zones, and thereby detected, ina single device.

The present devices may optionally further comprise an absorbent zonethat acts to absorb excess sample after the sample migrates through thetest region. The absorbent zone, when present lies in fluid flow contactwith the test region. This fluid flow contact can comprise anoverlapping, abutting or interlaced type of contact. In an occasionalembodiment, a control region (end of assay indicator) is provided in theabsorbent zone to indicate when the assay is complete. In thisembodiment, specialized reagents are utilized, such as pH sensitivereagents (such as bromocresol green), to indicate when the fluid samplehas permeated past all of the test and control zones. Alternatively, theend of assay control region may be effected by applying a line ofsoluble ink on the test region after all of the test and control zones,and at the interface with the absorbent zone. In general, the liquidfront moving through the capture zone will solubilize the ink andtransfer it into the absorbent. The resulting color change will be seenin an observation window above the absorbent zone, signifying end ofassay. Thus, these types of control regions are not specific for aparticular analyte. Generally, the absorbent zone will consist of anabsorbent material such as filter paper, a glass fiber filter, or thelike.

In an occasional embodiment, the fluid sample must be processed ortreated prior to contact with the device to ensure accurate detection ofat least one of the multiple analytes of interest. In this embodiment, areagent, such as an extraction solution, may be used to prepare thesample. Alternatively, reagents can be added to the test device afterinitial contact with the fluid sample. For example, the sample isintroduced to the device, and thereafter a reagent, such as a developersolution, is added to complete the assay.

FIG. 1 provides various representative configurations of the presentlycontemplated devices. Although not specifically limited to dipstick typeassays, these configurations may be incorporated in a dipstick-typeassay among other types of immunoassay devices contemplated herein. Thearrow in each Figure indicates the general direction of fluid sampleflow after initial contact with the device. The boxes containingvertical lines and/or the diagonal lines depict control zones, and theboxes containing crosshatched lines depict test zones. The samplereceiving zone [1], the label zone [2] and the test region [3] are alsodepicted. The present disclosure is not intended to be limited to theconfigurations depicted in FIG. 1(A-J). These views are merely providedfor illustrative purposes. For example, the test and control zones arenot necessarily of the same shapes and sizes depicted in FIG. 1.Further, the sample receiving zone, the label zone and test region arenot necessarily presented to scale. FIG. 1A depicts a device having twotest zones and a single control zone, wherein the control zone issituated between the test zones within the flow path. FIG. 1B depicts adevice having three test zones and two control zones, wherein thecontrol zones are situated between the test zones within the flow path.Further, in FIG. 1B, the second test zone is situated between the twocontrol zones within the flow path. FIG. 1C depicts a device having fourtest zones and two control zones, wherein the control zones are situatedbetween the test zones within the flow path. Further, in FIG. 1C, thesecond and third test zones are adjacent without a control zones betweenthese zones. FIG. 1D depicts a device having two test zones and a singlecontrol zone, wherein the control zone is situated downstream of thetest zones within the flow path. FIG. 1E depicts a device having twotest zones and a single control zone, wherein the control zone issituated upstream of the test zones within the flow path. FIG. 1Fdepicts a device having four test zones and three control zones, whereinthe control zones are situated between the test zones within the flowpath in a alternating fashion. FIG. 1G depicts a device having five testzones and one control zone, wherein the control zone is situated betweenthe second to last and last test zones within the flow path. Further, inFIG. 1G, the first through fourth test zones are adjacent to one anotherwithout having control zones between each test zone. FIG. 1H depicts adevice having three test zones and one control zone, wherein the controlzone is situated between the second and third test zones within the flowpath. FIG. 1I depicts a device having four test zones and three controlzones (wherein two control zones are comprised of the samelabeled-control reagent and binding reagent specific pair (oralternatively, are comprised of motifs that become visible when the testzone is moist), but one control zone (as indicated by the diagonallines) is comprised of one distinct pair of labeled control reagent andcontrol zone binding reagent). FIG. 1J depicts a device having four testzones and three control zones (wherein one control zone is comprised ofthe same labeled-control reagent and binding reagent specific pair (oralternatively, are comprised of motifs that become visible when the testzone is moist), but two control zones (as indicated by the diagonallines) are comprised of one distinct pair of labeled control reagent andcontrol zone binding reagent). Frequently, when two types of controlzones or pairs of control zones reagents are used, each control zone maybe colored differently. Other device configurations within the scope ofthe present disclosure are contemplated. For example, although notdepicted, the present disclosure contemplates a device having one testzone and one control zone, wherein multiple analytes can be detectedwithin the test zone.

Those of skill in the art will recognize that a variety of direct andindirect assay formats may be employed in the present devices. In afrequent embodiment, a direct assay format is utilized. Direct assaysare exemplified by those that detect the presence of an antigen in asample, as well as those that detect the presence of an antibody in asample.

As provided above, particular devices of the present invention include asupport. The support in these devices provides a convenient platform forperformance of the assay. However, the composition and shape of thesupport are not critical and may vary. Occasionally, the support may becomprised of a plastic or nylon material.

In frequent embodiment, the present devices are in the form of adipstick. Generally, dipsticks of the present invention are functionallyanalogous to the lateral assays described herein excepting the method ofcontacting a fluid sample. In embodiments configured as a dipstick, thematrix and support will generally be located on one end of the dipstick.The configuration of such devices will allow the device to be dipped orcontacted with a fluid sample with one end of a matrix i.e., the samplereceiving zone. After contacting the fluid sample, the sample preferablymigrates in an axial flow path through the matrix from the samplereceiving zone to the label zones and test region. Alternatively, thedevices of the present invention may be shaped so that samples may beapplied to the device by means other than dipping, e.g., application ofcontrolled amounts of sample by pipettes or the like.

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

EXAMPLES

Exemplary Test Device for Distinguishing Influenza A from Influenza B

An immunoassay device for use in determining the presence of anddistinguishing between multiple analytes was constructed in accordancewith the present invention. In particular, an immunoassay dipstickcapable of diagnosing influenza and distinguishing between influenza Aand influenza B infection was prepared. Those of skill in the art arefamiliar with the general methods for preparation of immunoassaydipstick devices (see, e.g., U.S. Pat. No. 5,712,172) as well as otherlateral flow immunoassays. The exemplary device described herein maylikewise be so constructed.

The exemplary test device comprised a sample pad containing sampleconditioning reagents, a label pad containing monoclonal antibodyagainst Influenza A nucleoprotein covalently bound to red colored latexmicroparticles (Duke Scientific), a monoclonal antibody againstInfluenza B nucleoprotein covalently bound onto red colored latexmicroparticles, and an unrelated protein (in this example, biotinylatedBSA) bound to blue latex microparticles (Duke Scientific). The devicewas assembled, in order, as a sample pad in fluid communication with alabel pad in fluid communication with a test pad (observation zone) influid communication with an absorbent pad at the device end. The padswere laminated, using 3M adhesive, onto a Mylar backing, in sequence andin fluid communication to allow lateral flow from one pad to the next. Astrip of plastic material, comprising a cutout (“window”) over theobservation zone, was then laminated on top of the device leaving aportion of the sample pad exposed for sample application. This plasticcover served the purpose of securing the pads in place and inoverlapping contact with one another, so that fluid communication wasmaintained there between. Additionally, the plastic material wasselected to provide sufficient rigidity to the final device to allowconvenient handling by the user and to isolate the areas of the dipstickthat are moistened during the testing process.

The observation zone was made of a Highflow Plus nitrocellulose membranefrom Millipore laminated onto a white Mylar backing. Test and controlreagents were deposited, using standard techniques, onto the observationzone membrane in the form of 3 lines, each perpendicular to the flow ofsample and in order upstream to downstream as follows: anti-Flu Bmonoclonal antibodies against a second epitope of the Influenza Bnucleoprotein at a concentration of about 3 mg/ml in phosphate bufferwas deposited as the a first test zone; at about 3 mm downstream of thefirst test zone, a control zone was formed by depositing streptavidinlabeled BSA; and at about 3 mm downstream of the first control zone asecond test zone was formed by depositing an antibody against a secondepitope of the Influenza A nucleoprotein (again, at a concentration ofabout 3 mg/ml in phosphate buffer). Thus, the two test zones and onecontrol zone were configured as illustrated in FIG. 1A.

Once the test and control reagents were deposited and dried onto thenitrocellulose membrane, the membrane was rendered non-bibulous bytreatment with a blocking solution. As discussed above, non-bibulousflow allows the components of interest in the assay to move atsubstantially the same rate along the test strip without preferentialretention of such components. This facilitates flow of a sufficientquantity of labeled material across the test and control zones, suchthat positioning of the control line upstream of the second test linedoes not interfere with the effectiveness or reliability of the controlzone.

At the end of the test device, the observation zone is in fluid contactwith a pad of absorbent material, in this example, made of absorbentcellulosic paper (Whatman).

As is common in the industry, the concentration of each component wasoptimized during development of the test to allow the assay to reach therequired sensitivity while avoiding non-specific binding. For instance,the colored latex/anti-Influenza A antibody conjugate was dilutedserially and deposited onto label pads that were dried, assembled intofunctional strips, and tested with liquid samples of the viralnucleoprotein at concentrations ranging from 7.5 ng/ml to 100 ng/ml. Thelowest concentration of conjugate allowing detection of the 7.5 ng/mlsolution of the nucleoprotein was selected as optimal. The optimalconcentration of anti-Influenza B antibody/colored latex conjugate wassimilarly optimized. The optimum concentration of the controlprotein/colored latex conjugate was selected to provide a line clearlyvisible to the eye after normal incubation of the assay. To constructthe final product, the optimized label (conjugate) solutions were mixedtogether then applied to and dried on the label pad using standardtechniques.

The size/capacity of the absorbent pad was optimized to ensure that asufficient amount of sample liquid would move through the test strip andacross the two test and one control zones to product a visible, accuratesignal when as little as 7.5 ng/ml of Influenza A and 7.5 ng/ml ofInfluenza B nucleoproteins were present in the sample.

Test of Exemplary Device

To confirm that positioning of a control zone upstream of a test zonewould not compromise the accuracy of a device according to the presentinvention, two devices were created and compared to one another. Bothdevices were constructed as described above, except that each wasconstructed with only an Influenza A test zone (single analyte). In onedevice, the control zone was located upstream of the test zone and inthe second device the control zone was located downstream of the testzone. A sample of Influenza A nucleoprotein at a relatively lowconcentration (sufficient to generate a visible signal of about 0.030O.D. measured on an optical density scanner after a 10 minuteincubation) was prepared and applied to each device. FIG. 2 shows theratios between the O.D. values of the test zone and the control zone fordifferent volumes of sample for each of the two devices. Increasingsample volumes were used, ranging from 25 uL up to 300 uL, facilitatingidentification of the minimum sample volume required to generatesufficient signal for the assay to meet design specifications. The graphin FIG. 2 clearly shows that the ratios of the signals generated by thetest zone and control zone in the two devices are virtually identicalregardless of the position of the test zone relative to the controlzone.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims. Citation herein of publications ordocuments is not intended as an admission that any of the foregoing ispertinent prior art.

What is claimed is:
 1. A method for the detection of multiple nucleicacid analytes in a fluid sample, the method comprising: contacting adevice with a fluid sample suspected of comprising one or more nucleicacid analytes of interest, each nucleic acid analyte of interest boundto a haptenic complementary nucleotide sequence, the device comprising amatrix defining an axial flow path, the matrix comprising: i) a samplereceiving zone at an upstream end of the axial flow path that receivesthe fluid sample, ii) a label zone positioned within the axial flow pathand downstream from the sample receiving zone, said label zonecomprising a mobilizable signal producing component that binds eachnucleic acid analyte of interest bound to its haptenic complementarynucleotide sequence, iii) at least two test zones positioned within theaxial flow path and downstream from the label zone, wherein each testzone comprises a restraining component that restrains each nucleic acidanalyte of interest bound to its haptenic complementary nucleotidesequence, and iv) a control zone positioned within the axial flow pathand downstream from the label zone, the control zone comprising animmobilized member of a specific binding pair that binds with a controlreagent deposited on the matrix; and inspecting the at least two testzones for signal producing component captured by the restrainingcomponent at each of the at least two test zones, where presence of thesignal producing component at a test zone indicates presence of thenucleic acid analyte of interest bound to its haptenic complementarynucleotide sequence.
 2. The method of claim 1, wherein the mobilizablesignal producing component is selected from the group consisting of achemiluminescent agent, a particulate label, a colorimetric agent, anenergy transfer agent, an enzyme, a fluorescent agent and aradioisotope.
 3. The method of claim 1, wherein the mobilizable signalproducing component is a colored particulate label.
 4. The method ofclaim 1, wherein the label zone comprises a mobilizable signal producingcomponent comprised of different colored particulate labeled reagents.5. The method of claim 1, wherein the mobilizable signal producingcomponent is a fluorescent agent.
 6. The method of claim 1, wherein therestraining component in one test zone of said at least two test zonesis an antibody that binds a nucleic acid analyte of interest bound to ahaptenic complementary nucleotide sequence.
 7. The method of claim 1,wherein the immobilized member in the control zone comprises anantibody.
 8. The method of claim 7, wherein the antibody in the controlzone is specific for a labeled control reagent.
 9. The method of claim1, wherein the nucleic acid analyte of interest is associated with asexually transmitted disease.
 10. The method of claim 9, wherein thenucleic acid analytes of interest are for detection of herpes.
 11. Themethod of claim 1, wherein the device is in the form of a dipstick assaydevice.
 12. The method of claim 1, where inspecting the at least twotest zones for signal producing component comprises inspecting viadirect visual, non-instrument for presence of a signal detectable. 13.The method of claim 1, wherein said label zone comprising a mobilizablesignal producing component that binds the haptenic complementarynucleotide sequence.
 14. A method for the detection of multiple analytesin a fluid sample, the method comprising: contacting a device with afluid sample suspected of containing one or more analytes of interest,the device comprising a matrix comprising: i) a sample receiving zonethat receives the fluid sample, ii) a label zone downstream from thesample receiving zone, said label zone comprising a mobilizable signalproducing component that binds an analyte of interest, iii) at least twotest zones, wherein each of the at least two test zones comprises acomponent for restraint of a different labeled analyte of interest, andiv) two or more control zones, wherein at least one of the control zonesis positioned downstream of a test zone in the at least two test zones,and wherein at least one of the control zones is a reference zone toprovide an indication of the relative location of one or more of the atleast two test zones; and detecting one or more labeled analytes ofinterest restrained in the one or more test zones.
 15. The method ofclaim 14, wherein the device comprises three or more test zones.
 16. Themethod of claim 14, wherein each of the test zones contains animmobilized reagent capable of specifically binding a unique analyte.17. The method of claim 14, wherein each of the test zones contains animmobilized reagent capable of specifically binding a unique labeledreagent.
 18. The method of claim 14, wherein the mobilizable signalproducing component in the label zone is a fluorescent agent.
 19. Amethod for the detection of multiple analytes in a fluid sample, themethod comprising: contacting a device with a fluid sample suspected ofcontaining one or more analytes of interest, the device comprising amatrix comprising: i) a sample receiving zone that receives the fluidsample, ii) a label zone downstream from the sample receiving zone, saidlabel zone comprising a first labeled reagent and a second labeledreagent, each of said labeled reagents capable of binding an analyte toform a labeled analyte and mobilizable in the presence of fluid sample,iii) at least two test zones downstream from the label zone, whereineach of the at least two test zones comprises a component for restraintof a different labeled analyte of interest, and iv) two or more controlzones, wherein at least one of the control zones is positioneddownstream of a test zone in the at least two test zones, and wherein atleast one of the control zones is a reference zone to provide anindication of the relative location of one or more of the at least twotest zones; and inspecting the at least two test zones for labeledanalyte.
 20. The method of claim 19, wherein at least one of the controlzones contains a component for restraint that provides for theimmobilization of the first labeled reagent or the second labeledreagent.
 21. The method of claim 19, wherein each of the test zonescomprises an immobilized reagent capable of specifically binding aunique analyte in the one or more analytes of interest.
 22. The methodof claim 21, wherein the first labeled reagent and the second labeledreagent are fluorescent agents.